Structure-based phylogeny of polyene macrolide antibiotic glycosyltransferases

Antibiotic glycosyltransferases (AGts) attach unusual deoxy-sugars to aglycons so antibiotics can exert function. It has been reported that polyene macrolide (PEM) AGts have different evolutionary origin when compared with other polyketide AGts, and our previous analysis have suggested that they could be results of horizontal gene transfer (HGT) from eukaryotes. In this paper, we compared the structures of PEM AGts with structures of eukaryotes and other AGts, and then built models of the representative PEM AGts and GT-1 glycosyltransferases. We also constructed the Neighbor-Joining (NJ) trees based on the normalized Root Mean Square (RMS) distance, the Bayesian tree guided by structural alignments, and carried out analysis on several key conserved residues in PEM AGts. The NJ tree showed a close relationship between PEM AGts and eukaryotic glycosyltransferases, and Bayesian tree further supported their affinity with UDP-glucuronosyltransferases (UGTs). Analysis on key conserved residues showed that PEM AGts may have similar interaction mechanism such as in the formation of hydrogen bonds as eukaryotic glycosyltransferases. Using structure-based phylogenetic approaches, this study further supported that PEM AGts were the result of HGT between prokaryotes and eukaryotes.     

PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16

AIMS: To assess the antibiotic biosynthetic potential of Amycolatopsis sp. strain UM16 and eight other Amycolatopsis species. METHODS AND RESULTS: Amycolatopsis genomic DNA was screened by PCR for the glycopeptide, Type-II (aromatic) polyketide and ansamycin biosynthetic gene clusters. Amycolatopsis sp. strain UM16, which exhibits weak antitubercular activity, was shown to have the glycopeptide oxyB gene and the Type-II (aromatic) polyketide-synthase KSalpha-KSbeta tandem gene pair, but not the AHBA synthase gene. The ristocetin (glycopeptide) producer, Amycolatopsis lurida NRRL 2430(T), was shown to have the oxyB gene and the Type-II polyketide-synthase KSalpha-KSbeta tandem gene pair. Amycolatopsis alba NRRL 18532(T) was shown to have the glycopeptide oxyB gene and the AHBA synthase gene. Phylogenetic analyses using Amycolatopsis oxyB and KSalpha-KSbeta gene sequences were conducted. CONCLUSIONS: Amycolatopsis sp. strain UM16 appears to have the biosynthetic potential to produce glycopeptide and Type-II polyketide antibiotics, but not ansamycins. The potential to synthesize aromatic polyketides may be more widely distributed in Amycolatopsis than is currently recognized. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening is a very useful tool for rapidly identifying the biosynthetic potential of an antibiotic-producing actinomycete isolate. Advanced knowledge of the type of antibiotic(s) produced will allow appropriate methods to be selected for antibiotic purification.     

Molecular engineering approaches to peptide, polyketide and other antibiotics

Molecular engineering approaches to producing new antibiotics have been in development for about 25 years. Advances in cloning and analysis of antibiotic gene clusters, engineering biosynthetic pathways in Escherichia coli, transfer of engineered pathways from E. coli into Streptomyces expression hosts, and stable maintenance and expression of cloned genes have streamlined the process in recent years. Advances in understanding mechanisms and substrate specificities during assembly by polyketide synthases, nonribosomal peptide synthetases, glycosyltransferases and other enzymes have made molecular engineering design and outcomes more predictable. Complex molecular scaffolds not amenable to synthesis by medicinal chemistry (for example, vancomycin (Vancocin), daptomycin (Cubicin) and erythromycin) are now tractable by molecular engineering. Medicinal chemistry can further embellish the properties of engineered antibiotics, making the two disciplines complementary.     

Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various streptomyces species

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.     

Crystal structure of SsfS6, the putative C‐glycosyltransferase involved in SF2575 biosynthesis

The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C‐glycosyltransferase (C‐GT) that forms the C‐C glycosidic bond between the sugar and the SF2575 tetracycline‐like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT‐B fold wherein the TDP and docked putative aglycon are consistent with the overall C‐glycosylation reaction. As one of only a few existing structures for C‐glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C‐glycosylation. Proteins 2013; 81:1277–1282. © 2013 Wiley Periodicals, Inc.     

放线菌来源的糖肽类抗生素的药学机制研究进展

糖肽类抗生素具有较好的抑制革兰氏阳性细菌生长的活性,临床上广泛用于治疗革兰氏阳性细菌导致的严重感染性疾病,也被认为是对抗这类顽固性病原菌的最后一道防线。随着耐药菌的不断涌现,糖肽类抗生素的应用越来越受到限制。本文针对糖肽类抗生素的结构特征与药效关系、生物学活性和病原菌对于它们的耐药机制,以及糖肽类抗生素的生物合成机制及其结构的合成生物学改造等方面进行了概述。最后,对糖肽类抗生素在应用中面临的问题进行了展望。     

Characterization of two glycosyltransferases involved in early glycosylation steps during biosynthesis of the antitumor polyketide mithramycin by Streptomyces argillaceus

A 2580-bp region of the chromosome of Streptomyces argillaceus, the producer of the antitumor polyketide mithramycin, was sequenced. Analysis of the nucleotide sequence revealed the presence of two genes (mtmGIII and mtmGIVv) encoding proteins that showed a high degree of similarity to glycosyltransferases involved in the biosynthesis of various antibiotics and antitumor drugs. Independent insertional inactivation of both genes produced mutants that did not synthesize mithramycin but accumulated several mithramycin intermediates. Both mutants accumulated premithramycinone, a non-glycosylated intermediate in mithramycin biosynthesis. The mutant affected in the mtmGIII gene also accumulated premithramycin A1, which contains premithramycinone as the aglycon unit and a D-olivose attached at C-12a-O. These experiments demonstrate that the glycosyltransferases MtmGIV and MtmGIII catalyze the first two glycosylation steps in mithramycin biosynthesis. A model is proposed for the glycosylation steps in mithramycin biosynthesis.     

Biosynthesis of Polyketide Antibiotics by Various StreptomycesSpecies that Produce Actinomycins

A collection of actinomycin-producing Streptomycesstrains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned actgene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolorrevealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between the biosynthetic pathways of actinomycins and polyketides is discussed.     

Persistence and resistance as complementary bacterial adaptations to antibiotics

Bacterial persistence represents a simple of phenotypic heterogeneity, whereby a proportion of cells in an isogenic bacterial population can survive exposure to lethal stresses such as antibiotics. In contrast, genetically based antibiotic resistance allows for continued growth in the presence of antibiotics. It is unclear, however, whether resistance and persistence are complementary or alternative evolutionary adaptations to antibiotics. Here, we investigate the co‐evolution of resistance and persistence across the genus Pseudomonas using comparative methods that correct for phylogenetic nonindependence. We find that strains of Pseudomonas vary extensively in both their intrinsic resistance to antibiotics (ciprofloxacin and rifampicin) and persistence following exposure to these antibiotics. Crucially, we find that persistence correlates positively to antibiotic resistance across strains. However, we find that different genes control resistance and persistence implying that they are independent traits. Specifically, we find that the number of type II toxin–antitoxin systems (TAs) in the genome of a strain is correlated to persistence, but not resistance. Our study shows that persistence and antibiotic resistance are complementary, but independent, evolutionary adaptations to stress and it highlights the key role played by TAs in the evolution of persistence.     

Characterization of two glycosyltransferases involved in early glycosylation steps during biosynthesis of the antitumor polyketide mithramycin by <Emphasis Type="Italic">Streptomyces argillaceus</Emphasis>

A 2580-bp region of the chromosome of Streptomyces argillaceus, the producer of the antitumor polyketide mithramycin, was sequenced. Analysis of the nucleotide sequence revealed the presence of two genes (mtmGIII and mtmGIV?) encoding proteins that showed a high degree of similarity to glycosyltransferases involved in the biosynthesis of various antibiotics and antitumor drugs. Independent insertional inactivation of both genes produced mutants that did not synthesize mithramycin but accumulated several mithramycin intermediates. Both mutants accumulated premithramycinone, a non-glycosylated intermediate in mithramycin biosynthesis. The mutant affected in the mtmGIII gene also accumulated premithramycin A1, which contains premithramycinone as the aglycon unit and a D-olivose attached at C-12a-O. These experiments demonstrate that the glycosyltransferases MtmGIV and MtmGIII catalyze the first two glycosylation steps in mithramycin biosynthesis. A model is proposed for the glycosylation steps in mithramycin biosynthesis.     

ppGpp介导的抗生素胁迫应答机制研究进展

抗生素是由微生物在生长发育后期产生的次级代谢产物,具有杀死或抑制细菌生长的能力,因此被广泛应用于细菌感染的临床治疗。在长期的进化过程中,细菌采取多种方式应对环境中抗生素的威胁。除了广为人知的抗生素耐药性(resistance)之外,细菌还能对抗生素产生耐受性(tolerance)和持留性(persistence),严重影响抗生素的临床疗效。鸟苷四磷酸(guanosine tetraphosphate, ppGpp)和鸟苷五磷酸(guanosine pentaphosphate, pppGpp) (本文统称ppGpp)是细菌应对营养饥饿等不利环境时产生的"报警"信号分子,其能够在全局水平调控基因的表达,使细菌适应不利的环境。越来越多的研究表明,ppGpp与细菌应对抗生素胁迫密切相关。基于此,本文综述了细菌中ppGpp的合成与水解及其作用机制,并重点阐述了ppGpp介导抗生素胁迫应答的分子机制,以期为新型抗生素的开发提供新思路。     

Ancient Horizontal Gene Transfer from Bacteria Enhances Biosynthetic Capabilities of Fungi

Background

Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive.

Methodology/Principal Findings

We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA)-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication.

Conclusions/Significance

Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase.     

The biosynthesis of glycopeptide antibiotics—a model for complex,non-ribosomally synthesized,peptidic secondary metabolites

Glycopeptide antibiotics are a class of widely known natural compounds produced by Actinomycetes. Vancomycin, the first member of the glycopeptide family to be discovered, was described in 1955 and used as an antibiotic soon thereafter. During the past 50 years numerous contributions on the structure, mode of action, and therapeutic features of vancomycin have been published. Recently, there has been considerable progress in elucidating the biosynthesis of glycopeptide antibiotics by combining molecular biology and analytical chemistry methods. Here, we provide an overview of the current knowledge regarding biosynthetic glycopeptide assembly.     

Spatial Organization and Stoichiometry of N-Terminal Domain-Mediated Glycosyltransferase Complexes in Golgi Membranes Determined by Fret Microscopy

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.     

Characterization of the biosynthetic gene cluster for the oligosaccharide antibiotic, Evernimicin, in Micromonospora carbonacea var. africana ATCC39149

Evernimicin (EV) belongs to the orthosomycin class of antibiotics and consists of several modified L- and D-deoxysugars containing unusual orthoester and glycosyl linkages and two orsellinic acid groups, one that is halogenated. The EV biosynthetic gene cluster from Micromonospora carbonacea var. africana ATCC39149 was localized by hybridization to a dTDP-D-glucose 4,6-dehydratase probe and a 120-kb region containing the EV biosynthetic cluster and surrounding regions has been sequenced. BLAST analysis has identified a type I polyketide synthase for orsellinic acid biosynthesis as well as enzymes required for L- and D-deoxyglucose and D-deoxymannose synthesis. In addition, genes involved in glycosyltransfer and resistance were identified. Insertional mutations in several biosynthetic genes blocked EV production, indicating a role for these genes in EV biosynthesis.     

Experimental evolution and genome sequencing reveal variation in levels of clonal interference in large populations of bacteriophage φX174

Background

The first step of GPI anchor biosynthesis is catalyzed by PIG-A, an enzyme that transfers N -acetylglucosamine from UDP- N -acetylglucosamine to phosphatidylinositol. This protein is present in all eukaryotic organisms ranging from protozoa to higher mammals, as part of a larger complex of five to six 'accessory' proteins whose individual roles in the glycosyltransferase reaction are as yet unclear. The PIG-A gene has been shown to be an essential gene in various eukaryotes. In humans, mutations in the protein have been associated with paroxysomal noctural hemoglobuinuria. The corresponding PIG-A gene has also been recently identified in the genome of many archaeabacteria although genes of the accessory proteins have not been discovered in them. The present study explores the evolution of PIG-A and the phylogenetic relationship between this protein and other glycosyltransferases.

Results

In this paper we show that out of the twelve conserved motifs identified by us eleven are exclusively present in PIG-A and, therefore, can be used as markers to identify PIG-A from newly sequenced genomes. Three of these motifs are absent in the primitive eukaryote, G. lamblia. Sequence analyses show that seven of these conserved motifs are present in prokaryote and archaeal counterparts in rudimentary forms and can be used to differentiate PIG-A proteins from glycosyltransferases. Using partial least square regression analysis and data involving presence or absence of motifs in a range of PIG-A and glycosyltransferases we show that (i) PIG-A may have evolved from prokaryotic glycosyltransferases and lipopolysaccharide synthases, members of the GT4 family of glycosyltransferases and (ii) it is possible to uniquely classify PIG-A proteins versus glycosyltransferases.

Conclusion

Besides identifying unique motifs and showing that PIG-A protein from G. lamblia and some putative PIG-A proteins from archaebacteria are evolutionarily closer to glycosyltransferases, these studies provide a new method for identification and classification of PIG-A proteins.     

Versatility of polyketide synthases in generating metabolic diversity

Polyketide synthases (PKSs) form a large family of multifunctional proteins involved in the biosynthesis of diverse classes of natural products. Architecturally at least three different types of PKSs have been discovered in the microbial world and recent years have revealed tremendous versatility of PKSs, both in terms of their structural and functional organization and in their ability to produce compounds other than typical secondary metabolites. Mycobacterium tuberculosis exploits polyketide biosynthetic enzymes to synthesize complex lipids, many of which are essential for its survival. The functional significance of the large repertoire of PKSs in Dictyostelium discoideum, perhaps in producing developmental regulating factors, is emerging. Recently determined structures of fatty acid synthases (FASs) and PKSs now provide an opportunity to delineate the mechanistic and structural basis of polyketide biosynthetic machinery.     

Accumulation of a Bioactive Benzoisochromanequinone Compound Kalafungin by a Wild Type Antitumor-Medermycin-Producing Streptomycete Strain

Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain.